Antimicrobial activity of kaurane diterpenes against oral pathogens

Antimicrobial Activity of Kaurane Diterpenes against Oral Pathogens Sergio R. Ambrosioa, Niege A. J. C. Furtadob, Dione´ia C. R. de Oliveirac, Fernando B. da Costab, Carlos H. G. Martinsa, Tatiane C. de Carvalhoa, Thiago S. Portoa, and Rodrigo C. S. Veneziania,* a b c

Nu´cleo de Pesquisas em Cieˆncias Exatas e Tecnolo´gicas, Universidade de Franca, Franca, SP, Brazil. Fax: +55-16-37 11 88 78. E-mail: [email protected] Departamento de Cieˆncias Farmaceˆuticas, Faculdade de Cieˆncias Farmaceˆuticas de Ribeira˜o Preto, Universidade de Sa˜o Paulo, Ribeira˜o Preto, Brazil Departamento de Fı´sica e Quı´mica, Faculdade de Cieˆncias Farmaceˆuticas de Ribeira˜o Preto, Universidade de Sa˜o Paulo, Ribeira˜o Preto, Brazil

* Author for correspondence and reprint requests Z. Naturforsch. 63 c, 326Ð330 (2008); received October 5/November 13, 2007 Two kaurane diterpenes, ent-kaur-16(17)-en-19-oic acid (KA) and 15-β-isovaleryloxy-entkaur-16(17)-en-19-oic acid (KA-Ival), isolated from Aspilia foliacea, and the methyl ester derivative of KA (KA-Me) were evaluated against oral pathogens. KA was the most active compound, with MIC values of 10 μg mLÐ1 against the following microorganisms: Streptococcus sobrinus, Streptococcus mutans, Streptococcus mitis, Streptococcus sanguinis, and Lactobacillus casei. However, KA did not show significant activity against Streptococcus salivarius and Enterococcus faecalis, with MIC values equal to 100 and 200 μg mLÐ1, respectively. Our results show that KA has potential to be used as a prototype for the discovery of new effective anti-infection agents against microorganisms responsible for caries and periodontal diseases. Moreover, these results allow to conclude that minor structural differences among these diterpenes significantly influence their antimicrobial activity, bringing new perspectives to studies on the structure-activity relationship of this type of metabolites with respect to caries and periodontal diseases. Key words: Kaurane Diterpenes, Oral Pathogens, Antimicrobial Activity, Aspilia foliacea

Introduction Dental plaque is a biofilm consisting of microorganisms generally present on the tooth surface. This biofilm plays an important role in the development of dental caries and periodontal diseases (Koo et al., 2000). Oral Streptococci, which are commonly isolated from human teeth, are the main microorganisms responsible for these infectious diseases (Hirasawa and Takada, 2002). Extensive efforts have been made toward the search for antibacterial compounds that can be incorporated into dental products (Cai and Wu, 1996). Chlorhexidine, one of the most important antibacterial agents against oral pathogens (Decker et al., 2005), has gained the approval of the American Dental Association Council on Dental Therapeutics (Cai and Wu, 1996). However, various adverse effects such as teeth staining and increased calculus formation are associated with the current use of products containing this compound (Cai and Wu, 1996). Also, chlorhexidine is much less effective at reducing the levels of 0939Ð5075/2008/0500Ð0326 $ 06.00

Lactobacillus, which are strongly related to caries progression in human mouths (Featherstone, 2006). These drawbacks justify the research on new effective antibacterial compounds that could be employed in caries prevention. Natural products, especially those derived from higher plants, provide a rich source of novel and diverse antimicrobial agents (Cai and Wu, 1996; Koo et al., 2000; Rı´os and Recio, 2005). However, little is known about the potential of secondary plant metabolites against oral pathogens (Cai and Wu, 1996; Koo et al., 2000). Many reports have extensively shown that kaurane-type diterpenes exhibit several important biological properties, including antiparasitic effects (Batista et al. 2007; Da Costa et al., 1996), antispasmodic and relaxant actions on the smooth muscle (Ambrosio et al., 2006; Tirapelli et al., 2004), as well as analgesic and antiinflammatory activities (Okuyama et al., 1991; Paiva et al., 2002). Members of this class of diterpenes have been reported to have cytotoxic effects, (Hanson, 2006; Ghisalberti, 1997), antiproliferative action on tumour cell cultures (Mongelli

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S. R. Ambrosio et al. · Antibacterial Activity of Kaurane Diterpenes

et al., 2002), and significant antimicrobial activity against Gram-positive and Gram-negative bacteria and yeasts, including Bacillus subtilis (Ghisalberti, 1997; Slimestad et al., 1995), Staphylococcus aureus, Mycobacterium smegmatis (Ghisalberti, 1997; Mitscher et al., 1983), Saccharomyces cerevisiae, Escherichia coli, Cladosporium herbarum, Candida albicans (Ghisalberti, 1997), among others. Moreover, a previous screening using Brazilian plants against oral pathogens performed in our laboratories showed that plant extracts rich in kaurane diterpenes are very effective against these microorganisms. Our results pointed out that these metabolites are a promising source of new prototypes for the development of antibacterial agents against cariogenic microorganisms. Considering the activity of plant extracts rich in kaurane diterpenes against oral pathogens, the aim of this study was to evaluate the activity of kaurane diterpenes isolated from Aspilia foliacea, as well as that of a semi-synthetic derivative, against some oral microorganisms, including Streptococcus mutans, which is considered one of the primary causative agents of dental caries. Materials and Methods Plant material Aspilia foliacea (Spreng.) Baker was collected in Brazil by Walter Vichnewski at the GO-118 highway (km 11) in November 1995. The plant material was identified by Joa˜o Moreira dos Santos (Museu Paraense Emı´lio Goeldi, PA, Brazil). A voucher specimen (Vichnewski # 367) was deposited in the herbarium of the Instituto de Biologia, Universidade de Campinas, SP, Brazil. Extraction and isolation Underground parts of Aspilia foliacea were airdried at 40 ∞C. The powdered material (650 g) was exhaustively extracted by maceration at room temperature using chloroform (5 L) to give the crude extract (6.1 g) after solvent evaporation under reduced pressure. This extract was re-suspended in methanol/water (9 :1, v/v) and extracted with n-hexane, followed by extraction with dichloromethane, to give crude extracts (0.45 and 1.12 g, respectively) after solvent evaporation under reduced pressure. The n-hexane extract (0.45 g) was chromatographed over silica gel 60 (0.063Ð0.200 mm) using classic chromatography with n-hexane and in-

327

creasing amounts of ethyl acetate as eluents. This procedure yielded 40 fractions (50 mL each), which were grouped into six new ones (Fr-1 to Fr-6) after TLC analysis. A solid mass appeared in Fr-2 (130 mg), and compound 1 [ent-kaur-16(17)-en-19oic acid, KA, 70 mg] was obtained after washing the solid with methanol. The dichloromethane extract (1.12 g) was chromatographed over silica gel 60H (Merck, art. no. 7736) using vacuum liquid chromatography (VLC) (Pelletier et al., 1986) with increasing amounts of ethyl acetate in n-hexane. Solvent was removed under reduced pressure in a rotatory evaporator, and six fractions (Fr-1 to Fr-6, 150 mL each) were obtained. Fr-3 (150 mg) was fractionated by medium pressure chromatography (“flash” chromatography) (Still et al., 1978) using silica gel 60 (Merck, art. no. 9385, 0.040Ð0.063 mm) and isocratic n-hexane/ethyl acetate (4 :1) and 1% acetic acid as the mobile phase, furnishing compound 2 [15-β-isovaleryloxy-ent-kaur-16(17)-en-19-oic acid, KA-Ival, 8 mg]. Preparative thin layer chromatography [PTLC, silica gel PF254 , 1 mm thickness, Merck, art. no. 7730, n-hexane/ethyl acetate (3 : 2) and 1% acetic acid] of Fr-4 (60 mg) furnished a mixture (7 mg) containing the diterpenes 15-β-hydroxy-ent-kaur16(17)-en-19-oic acid (3) and 17-hydroxy-ent-kaur15(16)-en-19-oic acid (4, respectively). KA (1) (about 50 mg) was treated with CH2N2 in Et2O (Da Costa et al., 1996), yielding the respective C-19 methyl ester derivative (KA-Me, 5). This compound was purified by flash chromatography with n-hexane/ethyl acetate (9 :1) as the mobile phase, and identified by means of spectrometric analysis. The purity of the evaluated diterpenes 1, 2 and 5 was estimated by thin layer chromatography using different solvent systems, as well as 1H and 13C NMR analysis. We estimated that the purity of all the compounds evaluated against the microorganisms was in the range 95Ð98%. Antimicrobial assays The minimum inhibitory concentration (MIC) values of the diterpenes were determined in triplicate using the microdilution broth method (Andrews, 2001) in 96-well microplates. Standard strains from the American Type Culture Collection of the following microorganisms were used: Enterococcus faecalis (ATCC 4082), Streptococcus

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S. R. Ambrosio et al. · Antibacterial Activity of Kaurane Diterpenes

salivarius (ATCC 25975), Streptococcus sobrinus (ATCC 33478), Streptococcus mutans (ATCC 25275), Streptococcus mitis (ATCC 49456), Streptococcus sanguinis (ATCC 10556), and Lactobacillus casei (ATCC 11578). The samples were dissolved in DMSO (dimethyl sulfoxide) at 1 mg mLÐ1, and were then diluted in tryptone soya broth; concentrations ranging from 200 to 5 μg mLÐ1 were achieved. The final DMSO content was 4% (v/v), and this solution was used as negative control. The inoculum was adjusted for each organism, to yield a cell concentration of 5 · 105 colony forming units (CFU) mLÐ1. One inoculated well was included, to allow control of the adequacy of the broth for organism growth. One noninoculated well free of antimicrobial agent was also included to assure medium sterility. Chlorhexidine was used as positive control. The microplates (96-well) were incubated at 37 ∞C for 24 h. After that, resazurin (30 μL) in aqueous solution (0.02%) was added to the microplates to indicate the microorganism viability (Palomino et al., 2002). The MIC was determined as the lowest concentration of the compound capable of inhibiting the microorganism growth. For the determination of either the bacteriostatic or bactericidal activities, the entire volume of each well of all the incubated microplates was subcultured on blood agar at 37 ∞C for 24 h. The absence of viable cell growth indicated a bactericidal effect. The minimum bactericidal concentration was defined as the lowest concentration that enabled no growth on blood agar. Results and Discussion The chemical structures of the diterpenes studied in this work are presented in Fig. 1. The spectral data of compounds 1 and 5 (Da Costa et al., 1996), 3 and 4 (Yahara et al., 1974), and 2 (Schteingart and Pomilio, 1981) are in agreement with those published in the literature. Among the evaluated diterpenes, compound 1 (KA) displayed the highest antibacterial activity (Table I), with MIC values of 10 μg mLÐ1 for the following microorganisms: S. sobrinus, S. mutans, S. mitis, S. sanguinis, and L. casei. For S. salivarius and E. faecalis, KA was not significantly active against these microorganisms, displaying MIC values of 100 and 200 μg mLÐ1, respectively. The MIC values of the pure diterpenes 2 and 5 ranged from 50 to values higher than 200 μg mLÐ1 (Table I).

Fig. 1. Chemical structures of the kaurane diterpenes 1-5 from Aspilia foliacea.

Chlorhexidine was used as positive control, and its MIC values for each microorganism are described in Table I. In recent years, the search for new anti-infection natural compounds has been the aim of many research groups (Rı´os and Recio, 2005), since many bacterial pathogens have developed defense mechanisms and resistance against antimicrobial agents (Kuzma et al., 2007). Therefore, it is reasonable to search for natural products that have antiplaque properties and antimicrobial activity against oral pathogens (Koo et al., 2000). In this work, we report on a comparison among structurally similar kaurane-type diterpenes with respect to their antibacterial activity against the main microorganisms responsible for dental caries and periodontal diseases for the first time. Rı´os and Recio (2005) analyzed the past, present and future of medicinal plants in the search for anti-infection agents, and they concluded that pure compounds displaying MIC values lower than or equal to 10 μg mLÐ1 are very promising for the development of antibacterial drugs. In this sense, our results show that compound 1 has potential to be used as a prototype for the discovery of new effective anti-infection agents against microorganisms responsible for caries and periodontal diseases. The results of the in vitro assays for 1 and 5 (Table I) clearly show that the presence of the carboxy group (C-19) causes an important contribution to the antibacterial activity against oral pathogens, and this activity decreases drastically in the case of the C-19 methyl ester. The structural analy-

S. R. Ambrosio et al. · Antibacterial Activity of Kaurane Diterpenes

329

Minimum inhibitory concentration [μg mLÐ1 (μm)] Microorganism Enterococcus faecalis Lactobacillus casei Streptococcus mitis Streptococcus mutans Streptococcus sanguinis Streptococcus sobrinus Streptococcus salivarius

Chlorhexidine 0.4 0.05 0.05 0.05 0.4 0.05 0.1

(0.69) (0.09) (0.09) (0.09) (0.69) (0.09) (0.17)

1 200.0 10.0 10.0 10.0 10.0 10.0 100.0

(661.26) (33.06) (33.06) (33.06) (33.06) (33.06) (330.63)

2

5

* * 200.0 (496.81) 100.0 (315.98) * * * * 50.0 (124.20) * * * * *

Table I. In vitro antibacterial activity of the kaurane diterpenes 1, 2, 5 and chlorhexidine against oral pathogens.

* Inactive in the evaluated concentrations.

scarce. However, comparing the MIC values against oral pathogens displayed by KA with those of previously described other secondary metabolites (Cai and Wu, 1996; Cunha et al., 2007; Tsuchiya et al., 1994), we can conclude that the kaurane-type diterpenes have potential use in the further development of natural anti-caries and anti-periodontal agents. Considering these results, it is important to emphasize that more biological, toxicological and structure-activity relationship studies must be undertaken, and the action mechanism of these diterpenes should be unveiled.

sis of compounds 1 and 2 allow to conclude that the introduction of an ester group (isovaleryloxy) at C-15 significantly decreases the investigated activity. Mendoza et al. (1997) evaluated some kaurane diterpenes against Gram-positive bacteria. The results of their study showed that the introduction of a hydrophilic 3-β-OH group drastically reduces the antibacterial activity of these compounds. The marked differences in the MIC values of some structurally very similar kaurane diterpenes previously reported by Mendoza et al. (1997), as well as the MIC values found in our present study reveal the great importance of understanding the structure-activity relationships of these compounds, since minor structural alterations may improve their activities against several bacteria. To date, the reports on the antimicrobial activity of natural products against oral pathogens are

This study was supported by Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq).

Ambrosio S. R., Tirapelli C. R., Da Costa F. B., and De Oliveira A. M. (2006), Kaurane and pimarane-type diterpenes from Viguiera species inhibit vascular smooth muscle contractility. Life Sci. 79, 925Ð933. Andrews J. M. (2001), British Society for Antimicrobial Chemotherapy standardised disc susceptibility testing method. J. Antimicrob. Chem. 48, Suppl. S1, 5Ð16. Batista R., Humberto J. L., Chiari E., and De Oliveira A. B. (2007), Synthesis and trypanocidal activity of ent-kaurane glycosides. Bioorg. Med. Chem. 15, 381Ð391. Cai L. and Wu C. D. (1996), Compounds from Syzygium aromaticum possessing growth inhibitory activity against oral pathogens. J. Nat. Prod. 59, 987Ð990. Cunha L. C. S., Silva M. L. A., Furtado N. A. J. C, Vinho´lis A. H. C., Martins C. H. G., Filho A. A. S., and Cunha W. R. (2007), Antibacterial activity of triterpenes acids and semi-synthetic derivatives against oral pathogens. Z. Naturforsch. 62c, 668Ð672. Da Costa F. B., Vichnewiski W., and Herz W. (1996), Diterpenes and synthetic derivatives from Viguiera aspil-

lioides with trypanomicidal activity. Planta Med. 62, 557Ð559. Decker E. M., von Ohle C., Weiger R., Wiech I., and Brecx M. (2005), A synergistic chlorhexidine/chitosan combination for improved antiplaque strategies. J. Periodont Res. 40, 373Ð377. Featherstone J. D. B. (2006), Delivery challenges for fluoride, chlorhexidine and xylitol. BioMed Central Oral Health 6, S8. Ghisalberti E. L. (1997), The biological activity of naturally occurring kaurane diterpenes. Fitoterapia 68, 303Ð325. Hanson J. R. (2006), Diterpenoids. Nat. Prod. Rep. 23, 875Ð885. Hirasawa M. and Takada K. (2002), Susceptibility of Streptococcus mutans and Streptococcus sobrinus to cell wall inhibitors and development of a novel selective medium for S. sobrinus. Caries Res. 36, 155Ð160. Koo H., Gomes B. P. F. A., Rosalen P. L., Ambrosano G. M. B., Park Y. K., and Cury J. A. (2000), In vitro antimicrobial activity of propolis and Arnica montana against oral pathogens. Arch. Oral. Biol. 45, 141Ð148.

Acknowledgements

330

S. R. Ambrosio et al. · Antibacterial Activity of Kaurane Diterpenes

Kuzma L., Ro´zalski M., Walencka E., Ro´zalska B., and Wysokinska H. (2007), Antimicrobial activity of diterpenoids from hairy roots of Salvia sclarea L.: Salvipisone as potential anti-biofilm agent active against antibiotic resistant Staphylococci. Phytomedicine 14, 31Ð35. Mendoza L., Wilkens M., and Urzu´a A. (1997), Antimicrobial study of the resinous exudates and of diterpenoids and flavonoids isolated from some Chilean Pseudognaphalium (Asteraceae). J. Ethnopharmacol. 58, 85Ð88. Mitscher L. A., Rao G. S., Veysoglu T., Drake S., and Haas T. J. (1983), Isolation and identification of trachyloban-19-oic and (Ð)-kaur-16-en-19-oic acids as antimicrobial agents from the prairie sunflower, Helianthus annuus. J. Nat. Prod. 46, 745Ð746. Mongelli E., Pomilio A. B., Sanchez J. B., Guerra F. M., and Massanet G. M. (2002), ent-Kaur-16-en-19-oic acid, a KB cells cytotoxic diterpenoid from Elaeoselinum foetidum. Phytother. Res. 16, 387Ð388. Okuyama E., Nishimura S., and Yamazaki M. (1991), Analgesic principles from Aralia cordata Thunb. Chem. Pharm. Bull. 39, 405Ð409. Paiva L. A., Gurgel L. A., Silva R. M., Tome A. R., Gramosa N. V., Silveira E. R., Santos F. A., and Rao V. S. (2002), Anti-inflammatory effect of kaurenoic acid, a diterpene from Copaifera langsdorffi on acid-induced colitis in rats. Vascul. Pharmacol. 39, 303Ð307. Palomino J. C., Martin A., Camacho M., Guerra H., Swings J., and Portaels S. (2002), Resazurin microtiter assay plate: simple and inexpensive method for detec-

tion of drug resistence in Mycobacterium tuberculosis. Antimicrob. Agents Chem. 46, 2720Ð2722. Pelletier S. W., Chokshi H. P., and Desai H. K. (1986), Separation of diterpenoid alkaloid mixtures using vacuum liquid chromatography. J. Nat. Prod. 49, 892Ð900. Rı´os J. L. and Recio M. C. (2005), Medicinal plants and antimicrobial activity. J. Ethnopharmacol. 100, 80Ð84. Schteingart C. D. and Pomilio A. B. (1981), Terpenoids from Wedelia buphthalmiflora. Phytochemistry 20, 2589Ð2590. Slimestad R., Marston A., Mavi S., and Hostettmann K. (1995), Larvicidal constituents of Melantheria albinervia. Planta Med. 61, 562Ð563. Still W. C., Kahn M., and Mitra A. (1978), Rapid chromatographic technique for preparative separations with moderate resolution. J. Org. Chem. 43, 2923Ð 2925. Tirapelli C. R., Ambrosio S. R., Da Costa F. B., Coutinho S. T., De Oliveira D. C., and De Oliveira A. M. (2004), Analysis of the mechanisms underlying the vasorelaxant action of kaurenoic acid in the isolated rat aorta. Eur. J. Pharmacol. 25, 233Ð241. Tsuchiya H., Sato M., Iinuma M., Yokoyama J., Tanaka T., Takase I., and Namikawa I. (1994), Inhibition of the growth of cariogenic bacteria in vitro by plant flavanones. Experientia 50, 846Ð849. Yahara S., Ishida M., Yamasaki K., Tanaka O., and Mihashi S. (1974), Minor diterpenes of Aralia cordata Thunb: 17-hydroxy-ent-kaur-15-en-19-oic acid and grandifloric acid. Chem. Pharm. Bull. 22, 1629Ð1631.